Antioxidants in food are of interest for four major reasons: they can protect the food itself against oxidative damage, they can exert antioxidant effects in the human gastrointestinal tract, they can be absorbed and exert antioxidant effects in other body tissues, and they may be used in plant extracts, or as pure compounds, as therapeutic agents. Evaluation of each of these functions requires methods for characterization of antioxidant activity in vitro, as well as techniques that accurately measure oxidative damage in the human body and can be used to evaluate the effects of antioxidants in vivo. This article critically reviews the techniques that are currently available for in vitro and in vivo antioxidant characterization.
Peroxynitrite is a cytotoxic species that can be formed, among other mechanisms, by the rapid reaction of superoxide with nitric oxide. Peroxynitrite formation has been implicated in a wide range of neurodegenerative and chronic inflammatory diseases, as has the formation of hypochlorous acid by myeloperoxidase. There is considerable interest in the development of peroxynitrite scavengers as therapeutic agents. The thiol compound mercaptoethylguanidine has been suggested to fulfil this role since it has recently been shown to be not only a potent inhibitor of inducible nitric oxide synthase but also a scavenger of peroxynitrite. Indeed, it has been shown to be protective in some experimental models of circulatory shock and inflammation at plasma levels in the approximate range 100–300 μ M . One protein inactivated by peroxynitrite is the major inhibitor of serine proteinases in human body fluids, α 1 ‐antiproteinase. At high (250–1000 μ M ) concentrations, mercaptoethylguanidine was found to be effective in preventing peroxynitrite‐mediated tyrosine nitration and α 1 ‐AP inactivation. By contrast, lower concentrations of mercaptoethylguanidine (1–60 μ M ) enhanced the inactivation of α 1 ‐antiproteinase by peroxynitrite. At all concentrations tested (1–1000 μ M ), mercaptoethylguanidine decreased the inactivation of α 1 ‐antiproteinase by hypochlorous acid. We suggest that products of reaction of mercaptoethylguanidine with peroxynitrite or peroxynitrite‐derived products could cause damage to α 1 ‐antiproteinase, and possibly other proteins in vivo , whereas scavenging of hypochlorous acid by mercaptoethylguanidine could contribute to its anti‐inflammatory action in vivo . British Journal of Pharmacology (1999) 126 , 1646–1652; doi: 10.1038/sj.bjp.0702465
Words such as “antioxidant” and “oxidative stress” are difficult to define. The term “antioxidant” used in the literature is restricted to chain-breaking antioxidant inhibitors of lipid peroxidation. Food scientists frequently equate antioxidants to inhibitors of lipid peroxidation because they use antioxidants to prevent rancidity. Free radicals generated in vivo damage proteins, DNA, and other molecules in addition to lipids. A broader definition is: an antioxidant is any substance that when present at low concentrations compared to those of an oxidizable substrate delays or prevents oxidation of that substrate. Mechanisms of antioxidant action can include: removal of O2, scavenging reactive oxygen/nitrogen species or their inhibiting ROS/RNS formation, binding metal ions needed for catalysis of ROS generation, and up regulation of endogenous antioxidant defenses. This chapter emphasizes the importance of the source of stress and the target (“oxidizable substrate”) measured; but also mentions the cases that the definition does not include. When ROS/RNS are generated in vivo, many antioxidants come into play. The chapter discusses the antioxidants known, or proposed, to be important in aerobic organisms. There is outline on some approaches to the characterization of direct antioxidant activity and discussion on biologically relevant ROS/RNS—superoxide and hydrogen peroxide, hydroxyl radical, peroxyl radicals, lipid peroxidation, hypochlorous acid, heme proteins/peroxides, peroxynitrite, and singlet oxygen—measuring their scavenging, their reactions, some inhibitions on radical formation and other properties. Some approaches have been listed to help prove that a given compound act as an antioxidant in vivo, including two approaches for putative antioxidants. Specific assays are being developed to measure rates of oxidative damage to protein, DNA, and lipid. Steady-state and total body oxidative damage to these targets can now be approximated, providing a tool to examine the effects of “antioxidants” in vivo.
When living organisms first evolved on the Earth, they did so under an atmosphere containing very little O2, i.e. they were essentially anaerobes. Anaerobic micro-organisms still survive to this day, but their growth is inhibited and they can often be killed by exposure to 21% O2, the current atmospheric level. As the O2 content of the atmosphere rose (as a result of the evolution of organisms with photosynthetic water-splitting capacity), many primitive organisms must have died. Present-day anaerobes are presumably the descendants of those primitive organisms that followed the evolutionary path of "adapting" to rising atmospheric O2 levels by restricting themselves to environments that the O2 did not penetrate. However, other organisms began the evolutionary process of evolving antioxidant defence systems to protect against O2 toxicity. In retrospect, this was a fruitful path to follow. Organisms that tolerated the presence of O2 could also evolve to use it for metabolic transformations (oxidases, oxygenases and synthases such as nitric oxide synthase [NOS]) and for efficient energy production by using electron transport chains with O2 as the terminal electron acceptor, such as those present in mitochondria. Human mitochondria make over 80% of the ATP that we need, and the lethal effects of inhibiting this, e.g. by cyanide, show how important the mitochondria are. Mitochondrial defects contribute to the pathology of a wide range of diseases, as shown in subsequent chapters of this book.
This chapter defines the terms oxidative stress and oxidative damage, and explains how they can affect cell behaviour: proliferation, adaptation, injury, senescence, intercellular communication, and cell death by apoptosis, necrosis, parthanatos (activation of poly-ADP ribose polymerase), and other mechanisms such as pyroptosis. The role played by reactive species (RS) in apoptosis is detailed. The effects of reactive species (RS) on ion channels and ion levels are presented, especially for K+, Ca2+, iron ions, and copper ions. Methods to measure the liberalization of 'catalytic' iron and copper ions by oxidative stress are described. There is a detailed description of the mechanisms by which RS (including hydroxyl radicals, singlet oxygen, hypochlorous acid, and peroxynitrite) cause damage to DNA, how this can lead to mutations, and how cells counter this by repair mechanisms. The ways in which defects in DNA repair raise the risk of cancer development in diseases, such as xeroderma pigmentosum, Lynch syndrome, ataxia telangiectasia, and Cockayne syndrome are presented. Oxidative protein damage and lipid peroxidation are explored in detail, including the mechanisms that cells have evolved to deal with them. The cellular actions of end-products of lipid peroxidation (isoprostanes, aldehydes, cholesterol oxidation products etc.) are reviewed. Heat-shock proteins, the acute phase response, the proteasome, and autophagy are described in detail. Redox regulation in bacterial, yeast and animal cells is reviewed in detail, and its physiological relevance discussed, especially for kinases, phosphatases (including PTEN), Nrf2, NF-κB (particularly the role of ROS in regulating its activity), AP-1, and mitochondria-to-nucleus signalling.
We have evaluated the abilities of ferulic acid, (±) catechin, (+) catechin and (-) epicatechin to scavenge the reactive oxygen species hydroxyl radical (OH±), hypochlorous acid (HOCl) and peroxyl radicals (RO2).Ferulic acid tested at concentrations up to 5 mM inhibited the peroxidation of phospholipid liposomes. Both (±) and (+) catechin and (-) epicatechin were much more effective. All the compounds tested reacted with trichloromethyl peroxyl radical (CCl3O2) with rate constants > 1 × 106M−1s−1.A mixture of FeCl3-EDTA, hydrogen peroxide (H2O2) and ascorbic acid at pH 7.4, has often been used to generate hydroxyl radicals (OH.) which are detected by their ability to cause damage to the sugar deoxyribose. Ferulic acid, (+) and (±) catechin and (-) epicatechin inhibited deoxyribose damage by reacting with OH. with rate constants of 4.5 × 109M−1s−1, 3.65 × 109M−1s−1, 2.36 × 109M−1s−1 and 2.84 × 109M−1s−1 respectively. (-) Epicatechin, ferulic acid and the (+) and (±) catechins exerted pro-oxidant action, accelerating damage to DNA in the presence of a bleomycin-iron complex. On a molar basis, ferulic acid was less effective in causing damage to DNA compared with the catechins.A mixture of hypoxanthine and xanthine oxidase generates O2 which reduces cytochrome c to ferrocytochrome c. (+) Catechin and (-) epicatechin inhibited the reduction of cytochrome c in a concentration dependent manner. Ferulic acid and (±) catechin had only weak effects.All the compounds tested were able to scavenge hypochlorous acid at a rate sufficient to protect alpha-1-antiproteinase against inactivation. Our results show that catechins and ferulic acid possess antioxidant properties. This may become important given the current search for "natural" replacements for synthetic antioxidant food additives.Key Words: Catechinferulic acidpro-oxidantanti-oxidantlipid peroxidationhypochlorous acid
Cerebrospinal fluid from patients with neuronal ceroid lipofuscinoses (infantile or Santavuori-Haltia type and juvenile type or Batten's disease) has a higher concentration of non-protein-bound iron and lower antioxidant activity than that of controls. These changes may contribute to the pathology of the disorders.
